Luminescent lanthanide chelates have become a primary focus of investigation due to their highly unusual spectral properties (Gudgin-Dickson et al. (1995) Pharmac. Ther. 66:207-235; Selvin, P. R. (2002) Annu. Rev. Biophys. Biomol. Strutt. 31:275-302; and Hemmila et al. (2005) J. Fluoresc. 15:529-542). These molecules have been used in wide variety of biochemical assays, including, for example, medical diagnostics, drug discovery, and as imaging tools in cell biological applications. Luminescent lanthanide chelates are especially useful as non-isotopic alternatives to conventional organic fluorophores in the applications where high background fluorescence is an issue. The unusual spectral (i.e., sharply spiked peaks) and temporal (i.e., long lasting emissions) properties of the luminescent lanthanide chelates can allow for (i) ultra-high sensitivity of detection (ii) facile, simultaneous monitoring of several analytes in the same sample mixture, and (iii) more information to be obtained from a given individual analyte in a sample.
A lanthanide probe can contain, for example, an organic fluorophore and a caged, or chelated lanthanide. The fluorophore moiety acts as an antenna, or sensitizer, which absorbs the energy of the excitation light and transfers it to the lanthanide in a radiation-less fashion. The antenna is required to “pump,” or activate the metal, since the absorbance of the lanthanide moiety is very low. The antenna-to-lanthanide energy transfer occurs only over a short distance (on the order of a few angstroms), which generally requires that the two moieties be tethered together.
Temporal and spectral gating enables unusually sensitive detection of lanthanide emission even in samples containing significant short-lived auto-fluorescence (e.g., biological specimens or tissues). These compounds are therefore potentially useful in a wide variety of technical and biological tasks, such as tracing analysis, immunoanalysis, tissue-specific imaging, and detection of single molecules in living cells.
Development of new luminescent probes is challenging, since the transfer of energy from the antenna to the lanthanide is complex (a process not yet well understood) and very sensitive to subtle structural variations in the fluorophore. Another challenge is the necessity of combining three functional units within the same reporting probe: an antenna, a chelated lanthanide, and a cross-linking group (for attachment to the biomolecule of interest). This requires a complex synthetic strategy, eventually leading to compounds whose size often exceeds 1,000 Da.
Two commonly used classes of lanthanide chelates are diethylenetriaminepentaacetic acid (DTPA) and tetraethylenetetraminohexaacetic acid (TTHA). These chelates attach to 7-amino quinolones, which are known as DTPA and/or TTHA-cs124 derivatives. The advantage of these classes of compounds is their high quantum yield, high solubility in water, and the possibility of introducing chemical modifications in the fluorophore to spectrally optimize the transfer of energy to the lanthanide, and to enable the attachment of a cross-linking group. A number of methods for the conjugation of these chelates to biomolecules have been suggested. One of them is to use the dianhydride form of DTPA, in which one of the anhydrides modifies the amino group of the chromophore, while the other anhydride reacts with amino group of the biomolecule. Even though this approach is technically simple, it raises concerns about the side reactions (modification of other nucleophilic groups) due to the high reactivity of anhydrides. The second approach takes advantage of the conjugation of one of the DTPA anhydride groups with the cs124 moiety, followed by reaction of the remaining anhydride with the diamine. The unmodified amino group of the resulted adduct can then be converted to an amino-reactive isothiocyano or thiol-reactive groups. This mode of attachment of the cross-linking group weakens the retention of the lanthanide within the chelate by eliminating one ligating carboxylate, and it also reduces the brightness of the lanthanide (30 to 1,000%) due to the quenching effect of the additional coordinated water molecule. These factors restrict in vivo applications where high concentration of metal scavengers is an issue (e.g., intracellular imaging). Analogous derivatives of the fluorophore coumarine have been suggested and used in biophysical studies. However, compared to their quinolone counterparts, they are less bright and do not support terbium (Tb) luminescence.
The unique photon emission properties of lanthanide-based probes render them suitable for a wide variety of applications that require ultrasensitive detection of biomolecules. Progress in this field depends on the availability of efficient probes. The complexity of energy pathways in luminescent lanthanide chelates is not fully understood, leaving much room for improvement in their applications as labels for probes. The development of more efficient probes is highly desirable, because new more challenging applications have arisen (e.g., for the detection of rare pathogens in environmental samples and detection of single molecules in cells).